Recent evidence has implicated NF-kappaB/Rel factors in the regulation of cytokine-mediated apoptosis of epithelial cells. The PI has demonstrated that treatment of murine hepatocyte cell lines with TGF-beta1 leads to a reduction in NF-kappaB activity causing apoptosis. NF-kappaB inactivation was correlated with hypophosphorylation and enhanced stabilization of the NF- kappaB/Rel specific inhibitory protein IkappaB-alpha. Ectopic expression of c-Rel rescued TGF-beta1-induced apoptosis in these cells. Thus, inhibition of NF-kappaB activity by TGF-beta1 is necessary for apoptosis to occur. Here the PI will dissect the regulation of expression of NF-kappaB by TGF-beta1 and will test the hypothesis that aberrant expression of NF-kappaB contributes to neoplastic transformation and resistance to TGF-beta1 induced apoptosis of hepatocytes. The role of the IkappaB-alpha gene product and the regulation of its expression during TGF-beta1 induced apoptosis of murine hepatocytes (Aim 1), and of v-ras or v-raf-transformed rat liver epithelial cells (RLEs)(Aim 2), will be elucidated. Also, TGF-beta1 treatment of murine hepatocyte cell lines causes a rapid transient induction of NF-kappaB activity that precedes NF-kappaB downregulation at later times, which correlates with enhanced phosphorylation and degradation of the IkappaB-alpha protein. Thus, in Aim 3, the residues within the IkappaB-alpha gene product phosphorylated in response to TGF- beta1, as well as the transcriptional regulation of IkappaB-alpha gene, will be determined. Furthermore, the kinase complex mediating the TGF-beta1-induced phosphorylation of IkappaB-alpha will be characterized and the efficacy of combined treatment of TGF-beta1 and NF-kappaB blockers in the induction of apoptosis of murine hepatocytes will be tested. These studies will provide valuable information on the regulation of NF-kappaB activity by TGF-beta1, a novel pathway of signalling. Furthermore, these findings will provide important insights into the role of NF- kappaB/Rel factors in the control of apoptosis of normal and transformed hepatocytes of potential clinical relevance.